We have found that proteases can stimulate both erythrodifferentiation (ED) and cell multiplication in mouse erythroleukemia (MEL) and human leukemia K562 cells. In contrast to studies of previously reported inducers which have not led to an understanding of the mechanism of their action, studies of protease inducers offer a unique opportunity to understand the mechanism of induction of ED because: (1)\induction by proteases is due to their proteolytic action; (2)\the critical proteolytic step(s) are on the cell and probably not on components of the medium; (3)\the cellular site of these types of inducer can be determined since their structure can be localized by radiolabeling and their function can be assayed; (4)\the substrate(s) (possibly receptor(s)) and products of a protease inducer can be determined; and (5)\protease induction can be regulated by specific protease inhibitors and synergistically enhanced by several agents, e.g., hemin. The cellular site(s) of protease activity and metabolism and cellular protease substrates and products will be determined in protease inducer-sensitive and -resistant cell lines using cell extracts, radioiodination, polyacrylamide gel electrophoresis, and radioautography. Proteases stimulate cell yield and appear to commit MEL cells to terminal cell division (TCD). These contradictory results will be studied by a semi-solid medium "commitment" assay. A kallikrein-like protease activity and hemin levels are elevated in plasma of some patients with hemolytic anemia. Since kallikrein and hemin act synergistically to induce ED in vitro, plasma samples from such patients will be tested for their effects on MEL and K562 cells. Experiments to test the possibility that a protease can be an erythropoietic signal in vivo using MEL and K562 cells have been designed. If ED is induced in vivo it may also induce commitment to TCD. If so, the malignant potential of the cells should be reduced and a type of differentiation chemotherapy will have been achieved. Proteases and/or hemin induce a fetal-like pattern of hemoglobin (Hb) synthesis in MEL and K562 cells. Since a fetal pattern is seen in some patients with hemolytic anemia, the Hb pattern present will be determined in the in vivo experiments. The effects of protease induction on cell membrane potential and on cellular Ca2+ levels also will be determined. The knowledge gained by these studies eventually should be of use in the prevention, diagnosis, and/or therapy of developmental, malignant, and acquired diseases. (M)